Apoptosis is a fundamentally important process in normal development and homeostasis. Apoptosis is also known to play a role in many disease states. For example, apoptosis and defects in apoptotic pathways are believed to be particularly relevant to cancer, heart disease, stroke, Alzheimer's disease, ischaemia and autoimmune diseases. For example, cancers may result from a defect in the apoptosis pathway, even in the absence of an increased proliferation rate.
Anti-cancer drug candidates failing to induce apoptosis are likely to have decreased clinical efficacy, making apoptosis assays important tools for high-throughput drug screening. The development of apoptosis modulating drugs requires robust assays for determining the presence of apoptotic and/or dead cells. Although a number of methods are available in the prior art to identify and/or quantify the presence of apoptotic and/or dead cells in a biological sample, for example a blood sample or a biopsy sample, the methods each suffer from specific disadvantages.
A number of methods are known in the art for assaying apoptosis. Nucleic acid stains may be used to identify apoptotic cells in cell populations, taking advantage of the characteristic breakdown of the nucleus during apoptosis, which results in collapse and fragmentation of the chromatin, degradation of the nuclear envelope and nuclear blebbing, resulting in the formation of “micronuclei”.
Other methods used in the prior art include measurement of protease activity in cells, for example, measurement of the activity of effectors of the apoptosis pathway, such as caspase-3 and caspase-8. As a characteristic of apoptosis is the disruption of active mitochondria, mitochondrial stains may be used to identify apoptotic cells. Other apoptosis assays employ free radical probes or ion indicators to measure changes in apoptotic cells. One of the most commonly used assays of apoptosis is based on the use of the vascular anticoagulant annexin V. Annexin V is a phospholipid-binding protein that has a high affinity for phosphatidylserine. However, in viable cells, annexin V does not bind phosphatidylserine as it is located on the cytoplasmic surface of the cell membrane. In apoptotic cells, however, phosphatidylserine is externalised to the outer surface of the plasma membrane, where it can be bound by annexin V. However, the use of annexin V in assays for the detection of apoptosis suffers from a number of disadvantages. In particular, as binding of annexin V to phosphatidylserine is Ca2+ dependent, the choice of buffer used in assays is limited. This usually requires a change of buffer prior to a sample being assayed, which is not only time-consuming but can cause increased stress to cells and result in an increase in non-viable cells.
As well as being used in the detection methods, antibody based techniques are commonly used in separation techniques, such as in the separation of sub-populations of cells in particular samples. Magnetic particles are routinely used to fractionate cells within populations of viable cells of multiple lineages (commonly lymphocyte subsets, monocytes, dendritic cells, stem cells, tumour cells etc) from many different species (human, mouse, rat, non-human primates and others). Generally, to ensure high fractionation efficiency, selection of cell populations is through indirect means: typically one or more primary antibodies are allowed to bind to cells and the antibody-bound cells are pulled out using magnetic particles coupled to a secondary antibody or, if the primary antibodies are biotinylated, (strept)avidin. Moreover such techniques generally require to be performed, at least in part, at low non-physiological temperatures and/or in the presence of buffers that differ in composition from the culture medium that is most suitable to support the viability of the cells in question.
There is therefore a need for further assays which may be used for detecting the presence of non-viable cells in a sample, for example, for use in high throughput assays and simple separation techniques which overcome some of the problems of the prior art techniques, in particular for techniques which may be used with minimal perturbation of physiological conditions of cells, such as optimal culture conditions (since perturbation reduces viability).